T-DNA Free Gene Editing through Transient Suppressing POLQ in Plants

Abstract

Disclosed are method and vectors for the application of POLQ in creating T-DNA free gene editing in plants. The method of simultaneous silencing of POLQ during CRISP/CAS9 gene editing increased the T-DNA free gene edited TO tobacco seedlings ratio. The method impacts the application of CRISPR gene editing technology in vegetatively propagated crops such as fruit trees by preventing the introduction of foreign DNA during gene editing.

1 . A method for editing a genome of a plant cell, the method comprising contacting the plant cell with an  Agrobacterium  that comprises a first nucleic acid sequence that encodes an oligonucleotide for silencing POLQ in the plant cell and a second nucleic acid sequence that encodes a genome editing system for editing a gene of interest, wherein contacting is conducted under conditions that allow for the first and second nucleic acid sequences to be introduced into the plant cell and the genome is transformed without integration of T-DNA. 
     
     
         2 . The method of  claim 1 , wherein the plant cell comprises a cell of a non-seeded perennial plant or an annual plant. 
     
     
         3 . The method of  claim 1 , wherein the plant cell is in an explant, plant organ, or seed. 
     
     
         4 . The method of  claim 1 , wherein the  Agrobacterium  comprises  Agrobacterium tumefaciens.    
     
     
         5 . The method of  claim 1 , wherein the  Agrobacterium  has been modified by introduction of a plasmid comprising SEQ ID NO 17-18 or a fragment thereof, or a sequence comprising at least 95% identity therewith, wherein the plasmid comprises the first nucleic acid sequence and optionally, the second nucleic acid sequence. 
     
     
         6 . The method of  claim 1 , wherein the genome-editing system comprises a nuclease, wherein the nuclease introduces a single-strand DNA break or a double-strand DNA break. 
     
     
         7 . The method of  claim 3 , wherein the genome-editing technique comprises a TALEN, a ZFN, meganuclease, or a CRISPR/Cas system. 
     
     
         8 . The method of any of  claim 1 , wherein the genome editing system introduces a genetic modification into the gene of interest or regulatory element thereof. 
     
     
         9 . The method of  claim 1 , wherein the conditions comprise incubating the plant cell in a first callus induction media, a second callus induction media, and a shoot induction media. 
     
     
         10 . The method of  claim 9 , wherein the second callus induction media comprises kanamycin. 
     
     
         11 . A method for editing a genome of a tobacco plant cell, the method comprising contacting the tobacco plant cell with an  Agrobacterium  that comprises a first nucleic acid sequence that encodes an oligonucleotide for silencing POLQ and a second nucleic acid sequence that encodes a genome editing system for editing a gene of interest, wherein contacting is conducted under conditions that allow for the first and second nucleic acid sequences to be introduced into the tobacco plant cell and the genome is transformed without integration of T-DNA. 
     
     
         12 . The method of  claim 11 , wherein the tobacco plant cell is in an explant, plant organ, or seed. 
     
     
         13 . The method of  claim 11 , wherein the  Agrobacterium  comprises  Agrobacterium tumefaciens.    
     
     
         14 . The method of  claim 11 , wherein the  Agrobacterium  has been modified by introduction of a plasmid comprising SEQ ID NO 17-18 or a fragment thereof, or a sequence comprising at least 95% identity therewith, wherein the plasmid comprises the first nucleic acid sequence and optionally, the second nucleic acid sequence. 
     
     
         15 . The method of  claim 11 , wherein the genome-editing system comprises a nuclease, wherein the nuclease introduces a single-strand DNA break or a double-strand DNA break. 
     
     
         16 . The method of  claim 15 , wherein the genome-editing technique comprises a TALEN, a ZFN, meganuclease, or a CRISPR/Cas system. 
     
     
         17 . The method of  claim 11 , wherein the genome editing system introduces a genetic modification into the gene of interest or regulatory element thereof. 
     
     
         18 . The method of  claim 11 , wherein the conditions comprise a first condition, a second condition, and a third condition. 
     
     
         19 . The method of  claim 18 , wherein the first condition comprises incubating the tobacco plant cells in a first callus induction medium for 2 days under no light. 
     
     
         20 . The method of  claim 19 , wherein the first callus induction medium comprises MS salts, 30 mg/L sucrose, 0.5-1.0 mg/L benzylaminopurine, and 0.1-0.75 mg/L NAA. 
     
     
         21 . The method of  claim 18 , wherein the second condition comprises incubating the tobacco plant cells in a second callus induction medium for 7 to 10 days under light. 
     
     
         22 . The method of  claim 21 , wherein the second callus induction medium comprises 100 mg/L Timentin and 60 mg/L kanamycin. 
     
     
         23 . The method of  claim 18 , wherein the third conditions comprise incubating the tobacco plant cells in a shoot induction medium. 
     
     
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